Kinetoplast DNA (kDNA) is the mitochondrial DNA of trypanosomes, parasitic protozoa that cause major human disease. The major subject of our study is Trypanosoma brucei that causes African sleeping sickness, although related parasites with kDNA cause Chagas disease and Leishmaniasis. kDNA is an unusual structure, consisting of DNA circles that are interlocked into a giant network that has a structure like that of medieval chain mail. The network contains several thousand minicircles and a few dozen maxicircles. Our major interest is the replication of kDNA. Major steps in this process include the sequential release of monomeric minicircles from the network by a topoisomerase to form free minicircles, replication of the free minicircles via theta structures, reattachment of the progeny minicircles to the network, and ultimately segregation of the double-size network into two daughter networks. One major emphasis of this proposal, presented in Aim 1, is to study proteins involved in kDNA replication. Because of the unusual structure of kDNA and because it is essential for parasite viability, these proteins could be targets for anti- trypanosome chemotherapy. Another approach, presented in Aim 2, is to study the regulation of kDNA replication. Unlike mitochondrial DNAs in higher eukaryotes, kDNA replicates during only part of the cell cycle, nearly concurrent with the nuclear S phase. We will try to understand what controls the timing. Finally, in Aim 3, we will study the molecular basis of segregation of the progeny networks that are formed during kDNA replication.